@article{HaegerWirgesTanzmannetal.2023,
  author    = {Haeger, Gerrit and Wirges, Jessika and Tanzmann, Nicole and Oyen, Sven and Jolmes, Tristan and Jaeger, Karl-Erich and Sch{\"o}rken, Ulrich and Bongaerts, Johannes and Siegert, Petra},
  title     = {Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis},
  series = {Microbial Cell Factories},
  journal   = {Microbial Cell Factories},
  number    = {22},
  publisher = {Springer Nature},
  issn      = {1475-2859},
  doi       = {10.1186/s12934-023-02079-1},
  pages     = {Article number: 77 (2023)},
  year      = {2023},
  abstract  = {Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.},
  language  = {en}
}
@techreport{HaegerBongaertsSiegert2023,
  author    = {Haeger, Gerrit and Bongaerts, Johannes and Siegert, Petra},
  title     = {Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep)},
  pages     = {17Seiten},
  year      = {2023},
  language  = {de}
}
@techreport{SiegertBongaertsWagneretal.2022,
  author    = {Siegert, Petra and Bongaerts, Johannes and Wagner, Torsten and Sch{\"o}ning, Michael Josef and Selmer, Thorsten},
  title     = {Abschlussbericht zum Projekt zur {\"U}berwachung biotechnologischer Prozesse mittels Diacetyl-/Acetoin-Biosensor und Evaluierung von Acetoin-Reduktasen zur Verwendung in Biotransformationen},
  address   = {Aachen},
  organization    = {FH Aachen},
  pages     = {16 Seiten},
  year      = {2022},
  language  = {de}
}
@article{JanusAchtsnichtTempeletal.2023,
  author    = {Janus, Kevin Alexander and Achtsnicht, Stefan and Tempel, Laura and Drinic, Aleksaner and Kopp, Alexander and Keusgen, Michael and Sch{\"o}ning, Michael Josef},
  title     = {Influence of fibroin membrane composition and curing parameters on the performance of a biodegradable enzymatic biosensor manufactured from Silicon-Free Carbon},
  series = {Physica status solidi : pss. A, Applications and materials science},
  volume    = {220},
  journal   = {Physica status solidi : pss. A, Applications and materials science},
  number    = {22},
  publisher = {Wiley-VCH},
  address   = {Weinheim},
  issn      = {1862-6300 (Print)},
  doi       = {10.1002/pssa.202300081},
  pages     = {Artikel 2300081},
  year      = {2023},
  abstract  = {Herein, fibroin, polylactide (PLA), and carbon are investigated for their suitability as biocompatible and biodegradable materials for amperometric biosensors. For this purpose, screen-printed carbon electrodes on the biodegradable substrates fibroin and PLA are modified with a glucose oxidase membrane and then encapsulated with the biocompatible material Ecoflex. The influence of different curing parameters of the carbon electrodes on the resulting biosensor characteristics is studied. The morphology of the electrodes is investigated by scanning electron microscopy, and the biosensor performance is examined by amperometric measurements of glucose (0.5-10 mM) in phosphate buffer solution, pH 7.4, at an applied potential of 1.2 V versus a Ag/AgCl reference electrode. Instead of Ecoflex, fibroin, PLA, and wound adhesive are tested as alternative encapsulation compounds: a series of swelling tests with different fibroin compositions, PLA, and Ecoflex has been performed before characterizing the most promising candidates by chronoamperometry. Therefore, the carbon electrodes are completely covered with the particular encapsulation material. Chronoamperometric measurements with H2O2 concentrations between 0.5 and 10 mM enable studying the leakage current behavior.},
  language  = {en}
}
@phdthesis{Achtsnicht2020,
  author    = {Achtsnicht, Stefan},
  title     = {Multiplex-Magnetdetektion von superparamagnetischen Beads zur Identifizierung von Trinkwasserkontaminationen},
  doi       = {10.18154/RWTH-2020-12052},
  pages     = {144 Seiten},
  year      = {2020},
  abstract  = {Die qualitative und quantitative Detektion von Zielsubstanzen innerhalb einer w{\"a}ssrigen Probe ist f{\"u}r viele Fragestellungen von Interesse, etwa bei der Detektion von Kontaminationen in Trinkwasser in Krisensituationen. Hierbei ist es nicht nur wichtig, dass Pathogene m{\"o}glichst sensitiv detektiert werden k{\"o}nnen, sondern auch, dass die Analyse schnell erfolgt, um Betroffenen im Katastrophenfall z{\"u}gig sicheres Trinkwasser zu Verf{\"u}gung stellen zu k{\"o}nnen. Da bei einem solchen Szenario nicht von einer in der N{\"a}he befindlichen funktionierenden Laborinfrastruktur ausgegangen werden kann, ist es wichtig, dass die Messung direkt vor Ort erfolgen kann. Im Rahmen dieser Arbeit wurde untersucht, ob eine derartige Schnellanalytik mithilfe von superparamagnetischen Beads (MBs) und der magnetischen Frequenzmischtechnik m{\"o}glich ist. Dabei werden die MBs mit Hilfe von prim{\"a}ren Antik{\"o}rpern an die Zielsubstanz gebunden und mit sekund{\"a}ren Antik{\"o}rpern an die Poren-Oberfl{\"a}che eines Polyethylen-Filters fixiert (Sandwich-Immunoassay). So kann die Quantifizierung der Zielsubstanz auf eine magnetische Messung der immobilisierten MB-Marker zur{\"u}ckgef{\"u}hrt werden. Die magnetische Frequenzmischtechnik basiert auf der Anregung der Probe mit Magnetfeldern zweier verschiedener Frequenzen. Die durch die nichtlineare Magnetisierungsform der superparamagnetischen MBs entstehenden Mischfrequenzen werden typischerweise mithilfe einer zweistufigen Lock-in-Detektion analysiert (analoge Demodulation), die in einem Magnetreader als Handheldger{\"a}t realisiert wurde. Zus{\"a}tzlich zu dieser Technik wurde das Prinzip der direkten Digitalisierung des gesamten Antwortsignals mit anschließender Fourier-Analyse der erzeugten Mischfrequenzen experimentell umgesetzt, um die Amplituden und Phasen mehrerer Mischfrequenzen simultan zu erfassen. Eine M{\"o}glichkeit zur Sensitivit{\"a}tssteigerung ist die magnetische Aufkonzentration, indem vor der magnetischen Analyse eine Separation der MBs aus einem gr{\"o}ßeren Probenvolumen mittels magnetischem Feldgradienten durchgef{\"u}hrt wird. Zur Charakterisierung verschiedener kommerzieller MBs hinsichtlich ihrer magnetischen Separierbarkeit wurde ein Aufbau zur Messung ihrer magnetophoretischen Beweglichkeiten realisiert und ihre Geschwindigkeiten im Gradientenfeld mikroskopisch gemessen.Da eine Probe oftmals nicht nur auf eine einzige Zielsubstanz, sondern simultan auf mehrere verschiedene Pathogene hin untersucht werden soll, wurden verschiedene Ans{\"a}tze entwickelt und getestet, die einen solchen multiparametrischen magnetischen Immunoassay erm{\"o}glichen. Einerseits wurde eine r{\"a}umliche Separation der Bindungsbereiche f{\"u}r verschiedene Zielsubstanzen realisiert, die sequentiell ausgewertet werden k{\"o}nnen. Andererseits wurde die Unterscheidung von verschiedenen Zielsubstanzen anhand der Charakteristika der an sie gebundenen, verschieden funktionalisierten MB-Typen untersucht. F{\"u}r eine solche Unterscheidung wurde zum einen die Anregefrequenz der magnetischen Frequenzmischtechnik w{\"a}hrend einer Messung variiert. Damit konnte gezeigt werden, dass sich verschiedene MB-Sorten anhand der Phase ihrer Frequenzmischsignale voneinander unterscheiden lassen. Weiterhin wurde gezeigt, dass sich der Signalverlauf einer bin{\"a}ren Mischung zweier verschiedener MB-Typen als gradueller {\"U}bergang der Verl{\"a}ufe der beiden reinen MB-L{\"o}sungen ergibt. Eine weitere Analysemethode f{\"u}r einen multiparametrischen Immunoassay besteht darin, ein zus{\"a}tzliches einstellbares statisches magnetisches Offsetfeld zu verwenden. Hierf{\"u}r wurden mehrere Aufbauten auf Basis von Permanent- und Elektromagneten simuliert, konstruiert und charakterisiert. Mithilfe von Simulationen konnte gezeigt werden, dass eine auf diesem Verfahren beruhende Unterscheidung f{\"u}r MBs mit unterschiedlichen magnetischen Partikelmomenten m{\"o}glich ist. Als direkte Anwendung des hier entwickelten Magnetreaders in Zusammenspiel mit der digitalen Demodulation wurde ein magnetischer Assay gegen die B-Untereinheit des Choleratoxins in Trinkwasser mit einem niedrigen Detektionslimit von 0,2 ng/ml demonstriert.},
  language  = {de}
}
@article{AchtsnichtPourshahidiOffenhaeusseretal.2019,
  author    = {Achtsnicht, Stefan and Pourshahidi, Ali Mohammad and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim},
  title     = {Multiplex detection of different magnetic beads using frequency scanning in magnetic frequency mixing technique},
  series = {Sensors},
  volume    = {19},
  journal   = {Sensors},
  number    = {11},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {1424-8220},
  doi       = {10.3390/s19112599},
  pages     = {13 Seiten},
  year      = {2019},
  abstract  = {In modern bioanalytical methods, it is often desired to detect several targets in one sample within one measurement. Immunological methods including those that use superparamagnetic beads are an important group of techniques for these applications. The goal of this work is to investigate the feasibility of simultaneously detecting different superparamagnetic beads acting as markers using the magnetic frequency mixing technique. The frequency of the magnetic excitation field is scanned while the lower driving frequency is kept constant. Due to the particles' nonlinear magnetization, mixing frequencies are generated. To record their amplitude and phase information, a direct digitization of the pickup-coil's signal with subsequent Fast Fourier Transformation is performed. By synchronizing both magnetic beads using frequency scanning in magnetic frequency mixing technique magnetic fields, a stable phase information is gained. In this research, it is shown that the amplitude of the dominant mixing component is proportional to the amount of superparamagnetic beads inside a sample. Additionally, it is shown that the phase does not show this behaviour. Excitation frequency scans of different bead types were performed, showing different phases, without correlation to their diverse amplitudes. Two commercially available beads were selected and a determination of their amount in a mixture is performed as a demonstration for multiplex measurements.},
  language  = {en}
}
@article{FalkenbergVossBottetal.2023,
  author    = {Falkenberg, Fabian and Voß, Leonie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra},
  title     = {New robust subtilisins from halotolerant and halophilic Bacillaceae},
  series = {Applied Microbiology and Biotechnology},
  volume    = {107},
  journal   = {Applied Microbiology and Biotechnology},
  publisher = {Springer Nature},
  address   = {Berlin},
  issn      = {1432-0614},
  doi       = {10.1007/s00253-023-12553-w},
  pages     = {3939 -- 3954},
  year      = {2023},
  abstract  = {The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5\% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.},
  language  = {en}
}
@article{AchtsnichtToedterNiehuesetal.2019,
  author    = {Achtsnicht, Stefan and T{\"o}dter, Julia and Niehues, Julia and Tel{\"o}ken, Matthias and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim and Schr{\"o}per, Florian},
  title     = {3D printed modular immunofiltration columns for frequency mixing-based multiplex magnetic immunodetection},
  series = {Sensors},
  volume    = {19},
  journal   = {Sensors},
  number    = {1},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {1424-8220},
  doi       = {10.3390/s19010148},
  pages     = {15 Seiten},
  year      = {2019},
  abstract  = {For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated.},
  language  = {en}
}
@article{AchtsnichtSchoenenbornOffenhaeusseretal.2019,
  author    = {Achtsnicht, Stefan and Sch{\"o}nenborn, Kristina and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim},
  title     = {Measurement of the magnetophoretic velocity of different superparamagnetic beads},
  series = {Journal of Magnetism and Magnetic Materials},
  volume    = {477},
  journal   = {Journal of Magnetism and Magnetic Materials},
  number    = {1},
  publisher = {Elsevier},
  address   = {Amsterdam},
  issn      = {0304-8853},
  doi       = {10.1016/j.jmmm.2018.10.066},
  pages     = {244 -- 248},
  year      = {2019},
  abstract  = {The movement of magnetic beads due to a magnetic field gradient is of great interest in different application fields. In this report we present a technique based on a magnetic tweezers setup to measure the velocity factor of magnetically actuated individual superparamagnetic beads in a fluidic environment. Several beads can be tracked simultaneously in order to gain and improve statistics. Furthermore we show our results for different beads with hydrodynamic diameters between 200 and 1000 nm from diverse manufacturers. These measurement data can, for example, be used to determine design parameters for a magnetic separation system, like maximum flow rate and minimum separation time, or to select suitable beads for fixed experimental requirements.},
  language  = {en}
}
@article{RabehiGarlanAchtsnichtetal.2018,
  author    = {Rabehi, Amine and Garlan, Benjamin and Achtsnicht, Stefan and Krause, Hans-Joachim and Offenh{\"a}usser, Andreas and Ngo, Kieu and Neveu, Sophie and Graff-Dubois, Stephanie and Kokabi, Hamid},
  title     = {Magnetic detection structure for Lab-on-Chip applications based on the frequency mixing technique},
  series = {Sensors},
  volume    = {18},
  journal   = {Sensors},
  number    = {6},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {1424-8220},
  doi       = {10.3390/s18061747},
  pages     = {14 Seiten},
  year      = {2018},
  abstract  = {A magnetic frequency mixing technique with a set of miniaturized planar coils was investigated for use with a completely integrated Lab-on-Chip (LoC) pathogen sensing system. The system allows the detection and quantification of superparamagnetic beads. Additionally, in terms of magnetic nanoparticle characterization ability, the system can be used for immunoassays using the beads as markers. Analytical calculations and simulations for both excitation and pick-up coils are presented; the goal was to investigate the miniaturization of simple and cost-effective planar spiral coils. Following these calculations, a Printed Circuit Board (PCB) prototype was designed, manufactured, and tested for limit of detection, linear response, and validation of theoretical concepts. Using the magnetic frequency mixing technique, a limit of detection of 15 µg/mL of 20 nm core-sized nanoparticles was achieved without any shielding.},
  language  = {en}
}